Since the birth in 1978 of Louise BROWN, the first child born as a result of in vitro fertilization, few major changes have taken place in the in vitro fertilization method.
Ovarian stimulation is now conventionally practiced on account of the ease with which oocyte retrieval can be programmed. The different ovarian stimulation procedures, clomiphene citrate - hMG, hMG alone, FSH - hMG and, more recently, the agonists of LH-RH associated with hMG, have considerably increased the number of oocytes obtained per retrieval (from 2.5 to 3.5 on average to 8 to 10 at the present time). This increase in the number of oocytes obtained has led to a larger number of embryos. In view of the risk of a multiple pregnancy caused by the transfer of more than three embryos, most assisted reproduction centers have acquired freezing apparatus to preserve the embryos and permit their secondary transfer replacement. Such stimulatory procedures are very expensive owing to the quantities of hormones used and the increased number of examinations (hormone doses and ultrasonic examinations) needed in order to monitor follicle growth.
The fertilization technique per se which is conducted in a laboratory using a CO.sub.2 incubator, a laminar flow hood, an inverted microscope, represents a major initial investment (see M. D. DAMEWOOD: In Vitro Fertilization:Insurance and financial considerations. Assisted Reproduction Review 1: 38, 1991). In addition the manipulation times, which are long due to the complexity of the technique, there is the need to monitor parameters such as CO.sub.2, temperature and relative humidity in the incubator. This correspondingly increases the cost of the procedure. This cost, which is high, estimated at $ 6,500 per attempt in the U.S.A. and approximately 15,000 Francs in France, makes it difficult to extend and disseminate this technique.
In addition the results which, at the best fertilization centers, are always below those for natural fertilization, namely 20% pregnancy per stimulated cycle the high cost, and the risks which result directly from this type of treatment (hyperstimulation, multiple pregnancies, storage and manipulation of frozen embryos) have prompted government agencies in the so-called developed countries to take steps to curtail the extension of assisted reproduction or procreation centers. Such steps run counter, of course, to the interests of sterile couples.
The applicant's experiments in in vitro fertilization, which were launched in 1979, have made it possible to develop a new procedure, which will be described hereinafter:
I. Intravaginal culture (IVC).
Reference is made, first of all, to international application WO 87/02879 filed on 7 Nov. 1986 and published on 21 May 1987, on which was based my U.S. Pat. No. 4,902,286 and to international application WO 88/08280 filed on 2 May 1988, on which was based my U.S. Pat. No. 5,084,004 and published on 3 Nov. 1988. All the features disclosed in these two my aforesaid U.S. Patents, shall be deemed to form part of the present description.
It was observed, in the first place, that the fertilization technique per se could be considerably simplified. Thus, I demonstrated, using the "CIVETE" technique, in French, "culture intra vaginale et transfert embryonnaire" or intravaginal culture and embryo transfer, that the addition of 5% CO.sub.2 enriched air was not necessary to maintain good culture conditions. However, to avoid any disturbance in the culture medium, it was necessary to fill the tube completely, without any air. Similarly, to avoid any major disturbance to the pH of the culture medium, the sperm concentration (responsible for metabolic consumption) was substantially reduced, 10,000 to 20,000 motile spermatozoa/ml, whereas the concentration is at least 50,000 mobile spermatozoa/ml in the conventional technique. This technique necessites fewer manipulations and requires no change in the culture medium.
In the conventional in-vitro fertilization technique, denuding or removal of the cumulus masa is carried out using a pipette or a needle 18 to 30 hours after insemination. This procedure necessitates considerable skill and can be traumatic for the embryo. In the IVC technique, spontaneous denuding of the embryos occurs in over 80% of cases. Such spontaneous denuding corresponds to a spontaneous detachment of the cells of the cumulus that surround the corona (see RANOUX C., AUBRIOT F. X., DUBUISSON J. B., CARDONE FOULOT H., POIROT C., CHEVALLIER O.: A new in vitro fertilization technique: intravaginal culture. Fertil Steril 49: 654, 1988 and RANOUX C., SEIBEL, M. C.: New techniques in fertilization: Intravaginal culture and microvolume straw. J In Vitro Fert "Embryo Transfer 7": 6, 1990); it reflects the quality of the culture and that of the gametes. In the event of toxicity, due either to the sterilization means (ethylene oxide), or to the faulty preparation of the instruments when removing the oocytes, a mass of dense cells of elastic appearance surrounding the embryo, which makes it necessary to extract the same in order to assess its stage of development. The embryo in the case of mechanical denuding, using a needle or a pipette, has never attained a stage of development of more than two cells, with fragments present and the cells having an irregular, asymmetrical and abnormal appearance.
The IVC technique is simple, inexpensive and requires only a small incubator and a stereoscopic microscope placed beneath a small-sized laminar flow hood. It does not necessitate any major laboratory infrastructure, in particular a CO.sub.2 incubator. The IVC technique patient participation, hence is accompanied in a better psychological context than conventional IVF techniques. This technique also constitutes a physiological improvement, with fertilization taking place in the vagina at body temperature, with the thermal variations normally observed in the periovulatory period. Fertilization carried out intravaginally raises fewer ethical problems and presents a lower risk of a mix-up of gametes. Moreover, there is no risk of an incubator breakdown which is liable to jeopardize fertilization of several patients. Thanks to its simplicity and standardization, this technique does not necessitate a high level of technical training and can be reproduced easily, without the results being affected.
It has been demonstrated, moreover, that results obtained using the IVC technique, were equivalent to those for conventional in-vitro fertilization, 15% births being obtained per retrieval.
II. Natural cycle with programming of retrieval by hCG injection.
The ovarian stimulation experiment, which is based on several years of work, with the different procedures mentioned above, has not enabled any significant difference between these procedures to be observed in respect of birth percentages per attempt. These rates, even in the best teams, are still between 10 and 15%. This was one of the main reasons that prompted us to revert to the natural cycle. Another of these reasons was the progress achieved since the first "test-tube baby".
When the first natural cycle attempts were made, follicle growth was monitored by urine testing for estradiol (E 2) and LH, without ultrasonographic verification, whence the lack of accuracy regarding oocyte maturity and the optimal time for oocyte retrieval. Ultrasonographic scanning, in association with the advent of quick assaying the amounts of hormones present in the blood stream, for estradiol and LH, has made for greater accuracy in determining ovulation. The use of hCG in the stimulated cycles has permitted programming of follicle puncture 34 to 36 hours after the injection, thus avoiding oocyte retrieval at just any time of the day or night.
Finally, the advent of new methods of retrieval, coelioscopy being replaced by ultrasonographic scanning, more recently practiced vaginally using a vaginal probe, has permitted major technical progress. Such retrieval, with ultrasonographic scanning, does not generally necessitate anaesthesia and obviates the need for a surgical infrastructure. Use of such advances has made it possible to develop a simplified natural cycle procedure with retrieval programming using human chorionic gonadotropin hCG (see FOULOT H., RANOUX C., DUBUISSON JB., RAMBAUD AUBRIOT FX., POIROT C.: In vitro fertilization without ovarian stimulation: a simplified procedure applied in 80 cycles. Fertil Steril 52: 617, 1989).
Follow-up of follicle growth begins only at the 8th or 9th day of the cycle, depending on the normal length of the patient's cycle; the number of hormone tests is very limited, on average five per cycle with only two or three ultrasonographic examinations.
If the follicle size is greater than 18 mm with E2 greater than 180 mg/ml and without an LH peak, hCG is injected and the oocyte is removed 34 to 36 hours thereafter. If the diameter is greater than or equal to 18 mm and E2 greater than or equal to 180 mg/ml with the LH between 20 and 40 IU/l, the hCG is injected immediately and retrieval is carried out 24 hours after receiving of the blood test results. Finally, if the LH exceeds 40 IU/l, hCG is not injected and the retrieval is carried out the day after the test.
This simplified procedure has made it possible to obtain, in a study of 80 attempts, a pregnancy rate of 22.5% per cycle, 17.5% of these were continuing pregnancies with being delivered in good health. This procedure offers a number of advantages: monitoring of follicle growth is shortened, simple and far less costly. The retrieval of a single follicle is fast, simple and well tolerated by the patient. This technique does not necessitate anaesthesia, just simple preliminary medication. Owing to the absence of medication, shorter periods of technical operations, and a limited use of disposable instruments, the cost of the procedure is considerably reduced. As no stimulation takes place and only one oocyte is retrieved, there is no risk of hyperstimulation. The problems due to embryo freezing are obviated. The possibility of multiple pregnancies, with the attendant obstetric risk and neonatal mortality, is non-existent.